Project summary/Abstract CTCF and cohesin causally organize mammalian genomes into topologically associating domains (TADs) by folding chromatin segments into loops. Since two DNA loci preferentially interact inside a TAD, TADs critically regulate gene expression by regulating enhancer-promoter contacts. Consistent with their crucial role in genome folding and gene regulation, CTCF and cohesin sub-units are among the most frequently mutated proteins in human cancers and also play prominent roles in neurological disorders. Understanding how dysregulation of CTCF and cohesin causes dysregulation of chromatin looping and gene expression in disease first requires a deep mechanistic understanding of how CTCF and cohesin regulate looping under physiological conditions. Dr. Hansen has previously established mouse stem cell lines where CTCF and cohesin are endogenously tagged. He found using 2D super-resolution imaging that CTCF and cohesin form small co-localizing clusters in the nucleus. This observation raises the possibility that clusters of CTCF and cohesin hold together chromatin loops. During the K99 phase, Dr. Hansen will investigate this hypothesis in Aim 1 by elucidating the detailed 3D nuclear organization of CTCF and cohesin using 3D super- resolution imaging at unprecedented resolution and the mechanism of clustering using an orthogonal biochemical approach. Moreover, the dynamics of chromatin looping are currently unknown. To address this gap in our understanding, Dr. Hansen will set up a system to visualize chromatin looping in live cells during the K99 phase of Aim 2 and elucidate the dynamics of chromatin looping in stem cells. With this information and these developments in hand, Dr. Hansen will then perform mechanistic and functional studies in the R00 phase. First, Dr. Hansen will use stem cell differentiation, induced gene activation and acute depletion perturbation experiments to understand how the dynamics of chromatin looping are functionally regulated during the R00 phase of Aim 2. Second, he will build on his K99 work in Aim 3 to understand the function of CTCF and cohesin clusters. Dr. Hansen's long-term goal is to become an independent principal investigator at a research institution and to understand the molecular mechanisms underlying chromatin looping and how this is dysregulated in disease. To help him achieve this goal, Dr. Hansen will be guided by his mentors and Scientific Advisory Committee. Training in the mentored K99 phase will expand Dr. Hansen's skill-set to include 3D super- resolution imaging, stem cell differentiation, microscope building and deepen his knowledge of cohesin biology. Moreover, Dr. Hansen will improve his writing, teaching, mentoring and management skills during the K99 phase. Completion of the research and training will greatly facilitate Dr. Hansen's transition to independence and success as an independent investigator.